Photosynthesis Research 52: 69±73, 1997. 69 c 1997 Kluwer Academic Publishers. Printed in the Netherlands. Technical communication A routine method to determine the chlorophyll a, pheophytin a and -carotene contents of isolated Photosystem II reaction center complexes Camiel Eijckelhoff & Jan P. Dekker Department of Physics and Astronomy, Institute of Molecular Biological Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands; Author for correspondence Received 13 February 1997; accepted in revised form 28 March 1997 Key words: chlorophyll, pheophytin, Photosystem II, reaction center Abstract The most simple way in which the stoichiometry of chlorophyll a, pheophytin a and -carotene in isolated Photosystem II reaction center complexes can be determined is by analysis of the spectrum of the extracted pigments in 80% acetone. We present two different calculation methods using the extinction coef®cients of the puri®ed pigments in 80% acetone at different wavelengths. One of these methods also accounts for the possible presence of chlorophyll b. The results are compared with results obtained with HPLC pigment analysis, and indicate that these methods are suitable for routine determination of the pigment stoichiometry of isolated Photosystem II reaction center complexes. Abbreviations: Car ± carotene; Chl ± chlorophyll; HPLC ± high-performance liquid chromatography; Pheo ± pheophytin; PS ± Photosystem; RC ± reaction center Introduction content of the PS II RC complex, since the number and energies of bound chlorophylls seriously affect the The smallest unit of Photosystem II (PS II) capable of interpretations of functional studies of the PS II reac- primary charge separation is the D1±D2-cytochrome tion center, such as those on the primary energy and b-559 reaction center complex (Nanba and Satoh electron transfer reactions (van Gorkom 1995). 1987). This complex is structurally related to the well- In a previous report we analyzed several HPLC characterized RC complex of photosynthetic purple and spectroscopic methods for the determination of bacteria and binds Chl a,Pheoaand -Car molecules the pigment stoichiometry of the PS II RC (Eijckelhoff as co-factors involved in the primary energy and elec- and Dekker 1995) and concluded that in some reports tron transfer reactions. Despite intensive research, the the Chl to Pheo ratio has been underestimated to very exact number of bound pigment molecules per com- signi®cant extents. In particular some of the applied plex has for long been a matter of debate. Kobayashi spectroscopic methods were suggested to yield rather et al. (1990) and Gounaris et al. (1990) suggested that unreliable results. This is unfortunate, because gener- the PS II RC complex binds 6 Chl a,2Pheoaand ally spectroscopic methods are well-suited for routine 2 -Car molecules. Other groups, however, suggested analysis of PS II RC preparations (they are easy to that the PS II RC complex in its most pure form binds perform and do not require special equipment). An 4Chla,2Pheoaand 1 -Car molecules (Montoya additional advantage of spectroscopic methods is the et al. 1991) and that higher contents of Chl a and - relative ease by which PS II RC complexes prepared Car arise from contamination with the core antenna in different laboratories can be compared. protein CP47 (Chang et al. 1994; Pueyo et al. 1995). In this report, we describe a very simple spectro- It is important to solve the question of the pigment scopic method well-suited for routine analysis to deter- PIPS NO.:138356 (M) BIO2KAP *138356* pres751.tex; 25/06/1997; 15:12; v.7; p.1 70 mine the Chl a,Pheoaand -Car contents of PS II RC preparations from the room temperature absorption spectra of their extracts in 80% acetone. The results are in line with the conclusion of a number of recent detailed studies (Eijckelhoff et al. 1996; Zheleva et al. 1996; Kurreck et al. 1997) that the isolated PS II RC complex in its most stable and pure form binds 6 Chl a,2Pheoaand 2 -Car molecules. Experimental The aim of this work is to determine the concentrations of Chl a, Pheo a and -Car of PS II RC preparations from their extracts in 80% acetone. For each wave- length the absorption A of the extract is given by: Figure 1. Room temperature absorption spectra of HPLC-puri®ed A =(c )+ (c ) ; ; chl chl pheo pheo Pheo a (full line) and Pheo a obtained by adding 0.75 mM HCl c ) +( to puri®ed Chl a (dashed line) in 80% acetone. The spectra were car car; (1) normalized as described in the text. So, if the molar extinction coef®cients of the three pigments are known at three different wavelengths, the concentrations c of the three pigments in the extract can be determined. Because for some preparations contam- errors in the estimation of the Pheo a extinction coef- ination of Chl b can not always be totally excluded, we ®cients will have relatively large effects on the calcu- present an additional method including this pigment. lated Chl/Pheo ratio of the extract. We prepared Pheo The extraction procedure of PS II RC prepara- a by adding 0.75 mM HCl to puri®ed Chl a in 80% tions from spinach in 80% acetone was described in acetone and recorded the absorption spectrum. Fig- detail before (Eijckelhoff and Dekker 1995). Chl a, ure 1 (dashed line) shows the spectrum of the obtained Chl b,Pheoaand -Car were obtained from such fraction, which in view of the spectral shape almost extracts with > 99.9% purity by reversed phase HPLC exclusively consists of Pheo a. The spectrum of HPLC- with 100% methanol as mobile phase (Eijckelhoff and puri®ed Pheo a in 80% acetone is shown as well (Figure Dekker 1995). The peak fractions were collected, dried 1, full line). Both spectra are very similar. Neverthe- by ¯ushing with nitrogen gas and re-solubilized in less, in the spectrum of the acidi®ed fraction a very 80% acetone, after which their absorption spectra were small amount (<1%) of Chl a may be present, because recorded on a Cary 219 spectrophotometer.The spectra the absorption near 430±435 nm is slightly stronger in of Chl a, Chl b and -Car were normalized to published case of the acidi®ed fraction, whereas the absorption extinction coef®cients in 80% acetone (86.3 mM 1 near 410±415 nm is slightly reduced. The spectrum cm 1 at 663 nm for Chl a, 134.14 mM 1 cm 1 at of HPLC-puri®ed Pheo a in Figure 1 (full line) was 460 nm for Chl b and 140 mM1 cm 1 at 454 nm for normalized to the integrated intensity of the acidi®ed -Car ± data from Lichtenthaler 1987), based on which spectrum, which is based on the assumption that the we obtained extinction coef®cients of these pigments total absorption between 370 and 720 nm of the <1% in 80% acetone at all recorded wavelengths (see Eijck- contamination of Chl a will result in a very similar elhoff and Dekker 1995, for the spectra of Chl a and total absorption if this fraction is converted into Pheo -Car in 80% acetone after normalization according to a. The spectrum of Pheo a in Figure 1 (full line) can these extinction coef®cients). therefore be regarded as the spectrum of highly puri- In our previous report we also presented the spec- ®ed Pheo a in 80% acetone, carefully normalized to trum of Pheo a in 80% acetone after normalization the spectrum of Chl a in 80% acetone. We note that the according to the extinction coef®cients as determined extinction coef®cient at the peak of the Qy band of Pheo by Lichtenthaler (1987), but for the work described a (665 nm) calculated by this method is 51.95 mM 1 in this report we decided to redetermine these coef- cm 1, which is identical to the value given by Licht- ®cients. The reason for this is that small systematic enthaler (51.9 mM 1 cm 1). pres751.tex; 25/06/1997; 15:12; v.7; p.2 71 Table 1. Extinction coef®cients at selected wavelengths of chlorophyll a, pheo- phytin a, -carotene and chlorophyll b in 80% acetone, calculated as described in the text Pigment Extinction coef®cient (mM 1 cm 1) 663 535551 480 460 431 412 Chlorophyll a 86.30 0.000 1.52 2.43 95.99 77.85 Pheophytin a 50.16 7.579 4.58 3.86 19.22 112.57 -Carotene 0.46 1.049 120.84 135.88 100.82 64.21 Chlorophyll b 43.95 134.14 57.43 20.04 =+ : A + : A With the extinction coef®cients of Chl a,Pheoa cp 0020 663 132 505 535551 and -Car being known at all wavelengths between 370 : A 1 150 480 (7) and 720 nm, Equation (1) can now be solved by using three key wavelengths (or four if the concentration of = : A : A Chl b is to be estimated as well). The best result is cc 0 146 663 4 054 535551 : A obtained when at each of these wavelengths the total + 8 311 480 (8) absorption is dominated by a different pigment. For 1 Method 1 we chose for 431 nm (where the absorption In these equations, c is given in MandA in cm ; of Chl a dominates the total absorbance),412 nm (Pheo the suf®ces a, p, c and b stand for Chl a,Pheoa, -Car and Chl b, respectively. a absorption dominates), 480 nm ( -Car absorption dominates) and 460 nm (to get an idea on possible Chl b contamination), whereas for Method 2 we chose for 663 nm (Chl a absorption dominates), 535±551 nm Results and discussion (Pheo a absorption dominates) and again 480 nm ( - Car).